Protein Folding and Assembly - American Chemical Society

University of California, Santa Barbara, California 93106,. Center for Polymer Studies, Department of Physics,. Boston University, Boston, Massachuset...
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Acc. Chem. Res. 2006, 39, 635-645

Elucidating Amyloid β-Protein Folding and Assembly: A Multidisciplinary Approach DAVID B. TEPLOW,*,#,†,§ NOEL D. LAZO,#,⊥ GAL BITAN,#,† SUMMER BERNSTEIN,| THOMAS WYTTENBACH,| MICHAEL T. BOWERS,| ANDRIJ BAUMKETNER,| JOAN-EMMA SHEA,| BRIGITA URBANC,‡ LUIS CRUZ,‡ JOSE BORREGUERO,3 AND H. EUGENE STANLEY‡ Department of Neurology, David Geffen School of Medicine, Brain Research Institute, and Molecular Biology Institute, University of California, Los Angeles, California 90095, Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, Center for Polymer Studies, Department of Physics, Boston University, Boston, Massachusetts 02215, and Center for the Study of Systems Biology, School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30318 Received March 29, 2006

A strong causal link between Aβ and AD has been established through genetic studies showing that autosomal dominant forms of AD invariably involve increased production of Aβ or an increased Aβ42/Aβ40 concentration ratio. In vitro biophysical studies have revealed that Aβ42 forms fibrils at significantly higher rates than does Aβ40. Importantly, Aβ42 self-association produces structures that are more neurotoxic than homologous structures formed by Aβ40. The postulated central role of Aβ in AD has focused therapeutic strategies on the control of Aβ production or self-association. Aβ fibrils are formed by a small number of stacked, extended, ribbon-like β-sheets, each of which is formed by β-strands arranged perpendicular to the fibril axis. To * To whom correspondence should be addressed. E-mail: [email protected]. # David Geffen School of Medicine, University of California, Los Angeles. † Brain Research Institute, University of California, Los Angeles. § Molecular Biology Institute, University of California, Los Angeles. ⊥ Current address: Gustaf A. Carlson School of Chemistry and Biochemistry, Clark University, 950 Main St., Worcester, MA 01610. | University of California, Santa Barbara. ‡ Boston University. 3 Georgia Institute of Technology.

ABSTRACT Oligomeric, neurotoxic amyloid protein assemblies are thought to be causative agents in Alzheimer’s and other neurodegenerative diseases. Development of oligomer-specific therapeutic agents requires a mechanistic understanding of the oligomerization process. This is a daunting task because amyloidogenic protein oligomers often are metastable and comprise structurally heterogeneous populations in equilibrium with monomers and fibrils. A single methodological approach cannot elucidate the entire protein assembly process. An integrated multidisciplinary program is required. We discuss here the synergistic application of in hydro, in vacuo, and in silico methods to the study of the amyloid β-protein, the key pathogenetic agent in Alzheimer’s disease.

1. Introduction The amyloid β-protein (Aβ) is a peptide that is ubiqui-

tously and normally expressed in humans predominately in two forms, 40- and 42-amino acids in length (Aβ40 and Aβ42, respectively) (see Lazo et al.1 for a comprehensive review). Aβ fibrils are the principal protein component of the extracellular deposits (amyloid plaques) characteristic of Alzheimer’s disease (AD).

David B. Teplow is a Professor of Neurology and Director of the Biopolymer Laboratory, David Geffen School of Medicine at UCLA. Dr. Teplow’s work seeks to interface the physical and biological sciences to facilitate a mechanistic understanding of disease and the subsequent development of rational therapeutic strategies. Noel D. Lazo is an Assistant Professor of Chemistry and Biochemistry, Clark University, and Adjunct Assistant Professor, Department of Neurology, David Geffen School of Medicine at UCLA. Dr. Lazo’s interests include the structural pathobiology of amyloidoses and dermatologic diseases. 10.1021/ar050063s CCC: $33.50 Published on Web 07/22/2006

 2006 American Chemical Society

Gal Bitan is an Assistant Professor, Department of Neurology, David Geffen School of Medicine at UCLA. Dr. Bitan’s program seeks to develop therapeutic agents for Alzheimer’s and other neurologic diseases linked to aberrant protein assembly. Summer Bernstein is a postdoctoral fellow, Department of Chemistry and Biochemistry, University of California at Santa Barbara. Dr. Bernstein is interested in studying solution conformation retention of proteins upon ionization into the gas phase, including aggregates of amyloid proteins linked to Alzheimer’s disease and bovine spongiform encephalopathy (BSE; “Mad Cow disease”). Thomas Wyttenbach is an Associate Research Professor, Department of Chemistry and Biochemistry, University of California at Santa Barbara. His interests include the structure and solvation of biologically interesting systems. Michael T. Bowers is a Professor of Chemistry, Department of Chemistry and Biochemistry, University of California at Santa Barbara. Dr. Bower’s interests include protein misfolding and aggregation, G-quadraplex formation and stabilization by drug candidates, and structural analysis of macromolecules in solventfree environments. Andrij Baumketner is a postdoctoral fellow in the Department of Chemistry and Biochemistry, University of California at Santa Barbara. Dr. Baumketner’s research interests are in theoretical approaches to problems in chemical and biological physics. Joan-Emma Shea is an Assistant Professor, Department of Chemistry and Biochemistry, University of California at Santa Barbara. Dr. Shea’s interests include developing and applying the techniques of statistical and computational physics to the study of biological problems. Drs. Brigita Urbanc and Luis Cruz are senior research associates, Department of Physics, Boston University. Their research includes studies of biopolymer systems and involves the development and application of modern methods of statistical mechanics: series, Monte Carlo, and renormalization group. Jose Borreguero is a postdoctoral fellow at the Georgia Institute of Technology. Dr. Borregueuro’s graduate work focused on the computational physics of protein folding and assembly. H. Eugene Stanley is University Professor; Professor of Physics and Physiology, College of Arts and Sciences; Director, Center for Polymer Studies; and Professor of Physiology and Biophysics, School of Medicine, Boston University. Dr. Stanley’s interests include the structure of liquid water, statistical physics, and the computational physics of complex biological systems. VOL. 39, NO. 9, 2006 / ACCOUNTS OF CHEMICAL RESEARCH

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Elucidating Amyloid β-Protein Folding and Assembly Teplow et al.

understand how these complex structures form, we have sought to identify assembly intermediates of decreasing complexity, beginning with fibrils and culminating in the study of the Aβ monomer. In 1997, discovery of the penultimate fibril assembly intermediate, the protofibril, was reported.2 Relative to mature amyloid fibrils, which commonly are observed as long (micrometer length), straight, unbranched filaments of diameter ∼10 nm, protofibrils are short (e150 nm), flexible, narrow (5 nm) assemblies that often have a beaded morphology. Importantly, protofibrils are potent neurotoxins.3 Continuing in vitro studies have revealed ever-smaller Aβ assemblies, all of which are neurotoxic.4 An increasing recognition of the biological importance of small Aβ assemblies has come through studies in animals and humans. Evaluation of neuronal function in transgenic mice expressing Aβ has revealed neurological deficits prior to amyloid deposition, suggesting that “soluble” Aβ assemblies were neurotoxic. Subsequent studies in humans have shown that oligomeric forms of Aβ are detectable in the brain and cerebrospinal fluid and that the levels of one type of oligomer, termed Aβ-derived diffusible ligands, are an order of magnitude higher in AD patients than in age-matched controls. These results support the hypothesis that Aβ oligomers are the proximate neurotoxins in AD.5 If the oligomer hypothesis is true, development of therapeutic agents would be facilitated by a mechanistic understanding of Aβ monomer folding and oligomerization. Ironically, the process of oligomerization interferes with the study of oligomerization. In any solution population of Aβ, monomers exist in different conformational states. At Aβ concentrations at which binary or higher-order collisions occur in an experimentally observable time regime, conformational complexity is increased by monomer self-association, which produces a mixture of metastable, noncovalently associated oligomeric assemblies that eventually form fibrils. This makes the use of spectroscopic techniques that yield population-average data, including CD, FT-IR, or NMR, problematic. The noncovalence of the oligomer state prevents oligomer fractionation and quantitation through SDS-PAGE because of SDS-induced dissociation.6 Aβ has not been crystallized, precluding the use of X-ray diffraction methods. How then does one understand the initial phases of Aβ folding and assembly? We posit that solution of the Aβ assembly problem requires multiple disciplines and the contemporaneous integration of results produced from them. We discuss here our combination of in hydro, in vacuo, and in silico approaches and how this combination has provided insights into the Aβ assembly problem that heretofore were unobtainable.

2. In Hydro Studies In hydro studies of pure populations of full-length Aβ peptides are seminal because they allow determination of intrinsic features of Aβ assembly without confounding 636 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 39, NO. 9, 2006

variables associated with ex vivo (e.g., plasma, cerebrospinal fluid, or brain homogenates) or in vivo (neuronal) Aβ preparations. In hydro studies provide a standard to which results of high-resolution, non-population-based (singlemolecule or oligomolecular) methods, such as mass spectrometry or computational physics, may be compared and thus validated. 2.1. Determining the Aβ Oligomer Size Distribution. Following earlier work defining protofibrillar intermediates2,3 (section 1), we sought to determine whether preprotofibrillar, nonmonomeric intermediates existed. To do so, we employed the method of photoinduced crosslinking of unmodified proteins (PICUP) to “freeze” particular equilibrium states of Aβ.7 PICUP covalently stabilizes oligomers in solution, allowing quantitative determination of the oligomer size distribution using techniques including SDS-PAGE and size exclusion chromatography (SEC). PICUP was used to determine the initial oligomerization states of Aβ40 and Aβ42 (Figure 5, inset). Aβ42 formed pentamer/hexamer units (“paranuclei”, blue arrowhead) that self-associated to form higher-order, protofibril-like oligomers (green arrowhead). Aβ40 did not form paranuclei but rather existed as a mixture comprising predominately monomer, dimer, trimer, and tetramer.8 The unique ability of Aβ42 to form paranuclei offered an explanation for its strong linkage to AD.8 2.2. Probing Nucleation of Aβ Monomer Folding. The discovery of a quantized Aβ42 size distribution suggested that some quasi-stable conformation must exist; otherwise a probabilistic distribution of oligomer sizes would have been observed. Secondary structure analyses have shown that monomeric Aβ is largely, but not entirely, disordered, and a quasi-stable monomer fold has been reported in solution-state NMR studies.9 Aβ oligomerization thus may involve pre-existent folds or monomer folding processes occurring contemporaneously with peptide self-association. To examine this question, we coupled the techniques of limited proteolysis and mass spectrometry. This approach has proven useful in the study of conformational changes in proteins that have a strong propensity to aggregate. Brief endoproteolysis is done under nondenaturing conditions at low enzyme/substrate ratios. Peptide mapping reveals protease-resistant protein segments that by inference must exist in the protein interior or possess stable folds. Using a panel of seven endoproteinases, we defined the temporal order of cleavages within monomeric Aβ40 and Aβ42.10 Four important results emerged: (1) the cleavage sites of both peptides were identical within the region Asp1-Val39; (2) the Val39-Val40 peptide bond was labile in Aβ40 but not in Aβ42; (3) the Val40-Ile41 peptide bond in Aβ42 was protease sensitive only under denaturing conditions; (4) a contiguous ten-residue region extending from Ala21 to Ala30 was protease resistant in both peptides. Observations 1-3 have relevance to and are consistent with the fact that the longer Aβ alloform, Aβ42, is linked particularly strongly to AD. Both alloforms have identical primary structure within the Asp1-Val40 region; thus it would be reasonable to predict that identical folding could

Elucidating Amyloid β-Protein Folding and Assembly Teplow et al.

FIGURE 1. Stereoviews of NMR-derived structural models of Aβ(21-30). Heavy-atom representations are shown with Glu22 (red), Val24 (gray), and Lys28 (blue) highlighted. Other atoms are black. All structures display a main chain turn at Val24-Lys28 and a relatively ordered N-terminus. The two families differ in the orientation of the Lys28 side chain. occur within this region, producing identical results in peptide mapping studies. Identical cleavages were observed within the first 39 residues.10 In contrast, differences in protease sensitivity might be observed if the Ile41-Ala42 dipeptide contributed to formation of an Aβ42-specific fold involving the peptide C-terminus. Observations 2 and 3 are consistent with the existence of such a postulated fold. The observation (no. 4) that the Ala21-Ala30 region in both Aβ peptides was protease-resistant suggested that this region was structured and might be the folding nucleus of the Aβ monomer.10 Peptidic forms of the folding nuclei of some globular proteins have been found to be stable and possess the same structure found in the cognate full-length protein. Indeed, we found that the Aβ(21-30) decapeptide displayed protease resistance identical to that of full-length Aβ.10 To determine the structure of Aβ(21-30), solution-state NMR studies were performed, yielding a structural model in which a primary motif was a turn formed by residues Val24-Gly25-Ser26-Asn27-Lys28 (Figure 1). The turn was stabilized by long-range Coulombic interactions between Lys28 and either Glu22 or Asp23 and hydrophobic interaction between the isopropyl and n-butyl side chains of Val24 and Lys28, respectively. The intrinsic propensity of the glycine-serine-asparagine residues to be involved in β-turns also could contribute to the favorable energetics of turn formation in the Val24Lys28 region. These data supported a hypothesis that turn

formation nucleated the intramolecular folding of the Aβ monomer. Interestingly, amino acid substitutions at Glu22 and Asp23 are linked to familial forms of AD and cerebral amyloid angiopathy.1 The turn model suggests that these substitutions cause disease through direct effects on Aβ monomer nucleation.

3. In Vacuo Studies The approaches discussed in sections 2.1 and 2.2 provided valuable information about low-order oligomerization and population-average monomer structure. However, PICUP is not 100% efficient, and therefore it progressively underrepresents oligomer frequency as order increases. Higherorder oligomers are unresolvable by SDS-PAGE. Limited proteolysis identifies flexible versus folded domains but reveals little about fold structure. A method able to determine oligomer size at high resolution in complex mixtures and to integrate with computational techniques of structure determination is ion mobility spectrometry (IMS).11 IMS can be conceptualized as an in vacuo analogue of SEC or gel electrophoresis, methods in which molecules of different size, under the influence of a constant fluid flow or electric field (E), respectively, move through matrices of defined porosity at different rates. In IMS, the matrix is helium gas in a drift tube. In the tube, ions are accelerated by a constant E and decelerated by collisions with He. The result is a constant drift velocity, VOL. 39, NO. 9, 2006 / ACCOUNTS OF CHEMICAL RESEARCH 637

Elucidating Amyloid β-Protein Folding and Assembly Teplow et al.

νD, that depends on E and a mobility constant K, according to eq 1.

νD ) KE

(1)

E and mass spectrometer geometry are known; therefore measurement of ion arrival time at the detector determines νD and, in turn, K. The special value of the IMS approach for studies of protein structure and assembly emanates from the dependence of K on the parameter σ, the [ion] collision cross section (the IMS equivalent of a Stoke’s radius in gel permeation chromatography). This relationship is expressed in eq 2.

K)

( )

3q 2π 16N µkBT

1/21

σ

(2)

Here q is ion charge, N is number density of helium gas, µ is reduced mass of the ion-neutral (He) complex, kB is Boltzmann’s constant, and T is temperature. Because σ depends on the shape of the ion and oligomer order, it is a key experimental constraint in computational modeling of ion structure (section 4.2). The most powerful feature of IMS is its ability to resolve ions of different mass m but identical m/z values, where z is charge. These ions are typical of amyloid assembly, in which homotypic self-association/dissociation can be described by eq 3, (n+1)q Mq + nMq T M(n+1)

(3)

where a variable number n of monomers, each of mass M and charge q, add to an initial monomer to produce an oligomer of order n + 1 carrying a charge (n + 1)q. A dimer M2 of charge 2q and a trimer M3 of charge 3q have m/z values identical to that of the monomer Mq; thus their mass spectra are identical, that is, their peaks are superimposed. However, in IMS,11 proteins almost always obey the relationship σn < nσ, where σn is the collision cross section of an nth-order oligomer. For example, σdimer is almost always smaller than 2σmonomer. Oligomers of identical m/z but different m, contributing to the same peak in the mass spectrum, thus can be resolved in the IMS experiment. The combination of MS and IMS allows determination of oligomer mass and shape and studies of self-association kinetics. In addition, thermodynamic characteristics of monomer and oligomer states can be examined in two ways, by the dependence of the arrival time on injection energy (through collision-induced decomposition (CID)) or temperature (Arrhenius analysis). 3.1. Monitoring Aβ Oligomerization. Bernstein et al.12 have shown that mass spectrometry of Aβ42 yields peaks with z/n of -4, -3, and -2 (Figure 2). Analysis indicates the -4 and -3 peaks come primarily from monomer (n ) 1) but the -2 peak comprises predominately oligomers (n > 1). In addition, a z/n peak of -5/2 is observed. This noninteger value indicates that the ions producing this peak are dimers or higher-order forms of Aβ. To characterize these multimers, the peak of z/n ) -5/2 was massselected and studied by IMS. Arrival time distributions (ATDs) were acquired using three different source ac638 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 39, NO. 9, 2006

FIGURE 2. Negative ion mass spectrum of Aβ42. The z/n values of the peaks are indicated.

FIGURE 3. Collision-induced decomposition of Aβ42 oligomers. ATD for the z/n ) -5/2 ion are shown with injection energies indicated. Letters designate dimer (D), tetramer (Te), hexamer (H), and dodecamer ((H)2). celeration voltages, producing three different injection energies, 23, 50, and 100 eV. Collision of ions with He atoms can produce energy-dependent conformational rearrangements of the ion to a more stable state, or in the case of multimeric species, ion dissociation. As shown in Figure 3c, relatively high injection energy (100 eV) yields an ATD with a major peak centered at ∼600-650 µs, a shoulder at ∼580 µs, and a minor peak centered at ∼350 µs. Because the selected ions were multimeric, it is reasonable to assign the dimer (D) state to the peak at ∼600-650 µs and expect that the shoulder and smaller, earlier peaks will contain higher-order species (section 3).

Elucidating Amyloid β-Protein Folding and Assembly Teplow et al.

Examination of the data at 50 eV (Figure 3b) is consistent with this expectation. Here three peaks clearly are visible at longer arrival times, at approximately 650, 580, and 480 µs. Beginning with the previously assigned -5 dimer at 650 µs, we assign the peak at 580 µs as the -10 tetramer (Te) and the peak at 480 µs as the -15 hexamer (H). The minor peak at 350 µs observed at 100 eV has a substantial magnitude and is assigned to the -30 dodecamer (H2). At low (23 eV) injection energy, little dissociation is observed (Figure 3a). The predominant ions are the hexamer (∼580 µs) and the dodecamer (∼360 µs). The observation of hexamer and dodecamer at low injection energies is significant for a number of reasons: (1) it suggests that IMS may overcome two major problems in understanding Aβ oligomerization, determining the oligomer size distribution quantitatively and monitoring changes in the distribution contemporaneously with higher-order assembly processes; (2) the identification of paranuclei by IMS-MS, a “noninvasive” approach without the chemical bias of PICUP, suggests that PICUP data for low-order oligomers are an accurate reflection of the oligomerization state; (3) observation of a hexamer T dodecamer equilibrium by IMS and PICUP supports the hypothesis that paranuclei form due to the natural propensity of the Aβ42 peptide to self-associate in a specific manner and that paranuclei assemble homotypically, not by monomer addition; (4) time-dependent formation of paranuclei8 and higher-order “oligo-paranuclei” have been observed by IMS-MS (Bernstein, S., in preparation), showing that study of the structural factors controlling oligomerization (section 2.2) and the effects of potential therapeutic agents on the process is feasible. 3.2. Thermodynamics of [Pro19]Aβ42 Oligomerization. In IMS, the drift environment is thermal, which allows measurement of the temperature dependence of gas-phase ion reactions that alter σ. If the rates k of these reactions obey the Arrhenius relationship, k ) A e-EA/kBT, in which kB is Boltzmann’s constant and T is temperature, the activation energy EA and the preexponential factor A can be determined. Recently, Bernstein et al.12 reported studies of [Pro19]Aβ42, an Aβ42 alloform containing a single amino acid substitution in the “central hydrophobic cluster” (CHC) region of the peptide, a region shown to be critical in the initiation and control of peptide assembly.13 [Pro19]Aβ42 displays limited high-order association relative to wild-type Aβ42,14 and IMS-MS experiments showed that this peptide forms monomers, dimers, trimers, and tetramers but not hexamers (paranuclei) or higher-order assemblies.12 Importantly, injection energy studies showed that ions comprising the -5/2 charge state underwent dimer (D) T tetramer (Te) transitions amenable to Arrhenius-type investigation. As seen in Figure 4c, D and Te exist in similar amounts at 300 K. With increasing temperature (Figure 4a,b), tetramer dissociation is evident. At higher temperature (440-510 K), dimer dissociation is observed (data not shown). Arrhenius analysis of the temperature dependence of the tetramer (k1) and dimer (k2) dissociation rates for the reaction k1

k2

Te 98 D 98 M yielded tetramer and dimer activation

FIGURE 4. Temperature dependence of [Pro19]Aβ42 multimer dissociation. ATD for the z/n ) -5/2 ion determined at an injection energy of 40 eV at the designated temperatures. Letters designate dimer (D) and tetramer (Te). energies of dissociation of 18.3 and 20.4 kcal/mol, respectively.12 It is noteworthy that the EA values determined by IMS-MS are similar to the 23 kcal/mol energy determined in hydro in quasielastic light scattering studies of Aβ40 monomer addition to the growing tip of the amyloid fibril.15 Two different methods thus suggest the same thing: substantial conformational rearrangement of the Aβ monomer is required for oligomerization and fibril elongation.

4. In Silico Studies In developing therapeutic agents for human diseases, it is useful to determine a target structure at atomic resolution. One of the most powerful methods to do so is computational (in silico) physics, the study of physical systems simulated in computers. In simulations of protein folding and self-association, the positions of every atom are known at each step, allowing determination of secondary, tertiary, and quaternary structure. Importantly, the effects of alterations in primary structure or simulation milieu (e.g., solvent polarity) can be determined. The in silico approach providing the most detailed information is “all-atom” molecular dynamics (MD) with explicit solvent. Here, all protein atoms are considered along with thousands of water molecules. Monitoring the positions and forces among thousands of atoms simultaneously and continuously is computationally demanding; thus the allatom MD approach is limited to time regimes of 10-20% R- or β-structure. This result was consistent with experimental data showing that freshly prepared Aβ is largely disordered in aqueous solution.29 The ability of REMD to reproduce experimentally observed σ values and secondary structure features in low-energy Aβ42 clusters suggests that expanded REMD studies of Aβ42-folding dynamics will be informative and relevant. 4.3. Folding of Aβ(21-30). 4.3.1. DMD Simulation. The earliest event in Aβ self-assembly is monomer folding. To study this process at high resolution, Borreguero et al.30 used DMD and a “united-atom” protein model (specifying 642 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 39, NO. 9, 2006

all atoms except hydrogens). Hydrogen bonding, electrostatic interactions, and solvent effects (implicit through hydropathic interactions) were implemented. Trajectories were produced at six different electrostatic interaction (EI) strengths, including those appropriate for cytoplasmic/ extracellular (aqueous; low EI) or membrane (lipid; high EI) milieus. Trajectories at zero EI strength were produced to account for a milieu in which electrostatic interactions are completely shielded by the solvent and to determine a relative contribution of EI to folding in other milieus. A representative structure from the simulations is shown in Figure 8. Key features include a global turn organization, hydrophobic interaction between the isopropyl side chain of Val24 and the n-butyl side chain of Lys28, electrostatic interactions between the N group of Lys28 and the carboxylates of Glu22 and Asp23, and a lack of backbone hydrogen bonds. Trajectories with nonzero EI displayed compaction of relatively extended conformers with concurrent decreases in the solvent-accessible surface area (SASA) and CR-CR distances of Val24 and Lys28. Hydrophobic interactions were the primary force driving turn formation. Electrostatic interactions stabilized the turn. These observations were consistent with experimental10 (section 2.2) and computational27 (section 4.1) results. The EI-dependence of turn structure was illuminating. The Lys28 side chain “flipped” from one side of the plane of the turn to the other, depending on EI. At low to moderate EI, Lys28-Glu22 electrostatic interactions were favored. At higher EI, the Lys28 side chain flipped to the other side of the turn, favoring Lys28-Asp23 interactions. The latter interaction has been shown to occur in fibrils. The data predict that mutations destabilizing Glu22-Lys28 interactions or stabilizing Asp23-Lys28 interaction could facilitate fibril formation and thus be pathogenic. With respect to the latter point, Sciarretta et al. have shown that covalent cross-linking (lactam formation) between Lys28Asp23 eliminates the lag phase in fibril formation and increases the fibril formation rate by a factor of ∼1000.31 In fact, all human disease-causing mutations affecting the Ala21-Ala30 region of Aβ appear to alter the stability of the turn (Grant et al., in preparation). 4.3.2. All-Atom MD Simulation. The relatively small number of atoms in the Aβ(21-30) system made it amenable to all-atom MD simulations in explicit water.

Elucidating Amyloid β-Protein Folding and Assembly Teplow et al.

FIGURE 9. Conformations within clusters C1 (a and b) and C2 (c and d). Centroids of the clusters are shown in panels a and c. Superimpositions of conformers are shown in panels b and d. Cruz et al.32 simulated five folding processes: (1) wild type (WT) Aβ(21-30) in “random coil” (RC) conformation; (2, 3) the average family I and II turn structures from Lazo et al.10 (Figure 1) in reduced density water; (4) Aβ(21-30) containing the Glu22 f Gln “Dutch” substitution; (5) WT peptide in high ionic strength water (containing NaCl). In all five trajectories, the conformers displayed relatively rigid turns with highly flexible termini, as seen experimentally in prior NMR studies.10 Hydrophobic events, characterized by packing of the Val24 isopropyl side chain with the Lys28 n-butyl side chain, predominated over electrostatic interactions involving the side chains of Glu22, Asp23, and Lys28. For the WT peptides, the hydrophobic and electrostatic interactions occurred simultaneously frequently (>70% of the time), consistent with the suggested stabilizing role of electrostatics.10 An observation highlighting the ability of single-molecule methods (in silico techniques) to reveal interactions that averaging methods (NMR among others) cannot is that of a periodic, close (9 Å) Coulombic interactions among these residues.10 In all trajectories except the “NaCl,” the Glu22-Lys28 and Asp23-Lys28 electrostatic interactions were mutually exclusive, consistent with the

flipping of the Lys28 side chain observed by Borreguero et al.30 In the trajectory with NaCl, contemporaneous Glu22-Lys28 and Asp23-Lys28 interactions and Val24Lys28 packing were observed, possibly due to salt effects on peptide-water hydrogen bonding. 4.3.3. REMD Simulation. All-atom REMD with explicit solvent also has been applied to the Aβ(21-30) folding problem.26 Two structural clusters were observed with occurrence frequencies g5%, C1 (30%) and C2 (10%). C1 occupied the global minimum on the free energy surface and C2 occupied a local minimum. The thermodynamic stability of these clusters suggested that their component conformers were biophysically relevant. Figure 9 shows the most representative conformation from C1 (panel a) and a superimposition of C1 conformers (panel b) to illustrate conformational variability. A stable core involving Glu22-Lys28 and displaying a bend between Val24 and Lys28 was observed. Lys28(Nζ)-Glu22(Cδ) distance measurements revealed two maxima (3.4 and 6.3 Å), suggesting the existence of short-range (salt-bridge) and longrange (water-mediated) Coulombic interactions. One longrange (∼6.5 Å) interaction was seen between Lys28(Nζ) and Asp23(Cγ) atoms. Interestingly, strong hydrogen bonds were noted between Asp23(Oδ) and Gly25, Ser26, Asn27, and Lys28. Hydrogen bonds were not seen by VOL. 39, NO. 9, 2006 / ACCOUNTS OF CHEMICAL RESEARCH 643

Elucidating Amyloid β-Protein Folding and Assembly Teplow et al.

NMR,10 possibly because the spectra are ensemble averages of peptide structures. C2 conformers also possess a bend (Figure 9, panels c and d), but the Glu22-Lys28 salt bridge is absent, and hydrogen bonding patterns differ significantly. An important difference between C1 and C2 is the position of the Lys28 side chain, which exists on opposite sides of the bend plane in the two clusters, as observed in NMR studies.10 A novel result of the REMD studies is insight into the unusual protease resistance of Aβ(21-30). One can compare C1 conformer structures to significantly (CR RMSD >1 Å) divergent structures in other clusters. Divergent conformers can be considered higher energy, non-native states analogous to denatured conformers, states predicted to have larger radii of gyration, molecular volumes, and SASA. Surprisingly, divergent conformers displayed only modest increases in these parameters (5.1 vs 4.5 Å, 937.9 vs 933.5 Å2, and 10.6 vs 10.0 Å, respectively), and all possessed the central Val24-Lys28 bend.26 Thus, in both the lowest-energy “native state” and higher-energy “denatured” states, the Aβ(21-30) peptide maintains its bend topology and overall size. This conformational stability may explain the extraordinary protease resistance of this region of Aβ and its lack of aggregation, consistent with its low propensity to fold into an aggregationcompetent conformation. 4.3.4. Simulating Aβ(21-30) Folding: A Synthesis. The greatest uncertainty in in silico studies is the level at which they reproduce physical reality. Confidence in the relevance of simulations comes from agreement among studies done using different algorithms and, importantly, from agreement with experiment. The three different Aβ(21-30) simulation approaches produced a consistent picture of an Ala21-Ala30 fold characterized by a turn or bend structure stabilized by hydrophobic and Coulombic interactions and displaying flexible termini. These structural models were consistent with results of biochemical, mass spectrometric, and NMR experiments (sections 2 and 3). For example, CR RMSD values between turn region models based on simulation26,30 and NMR-derived constraints10 were as low as 0.7-1.1 Å. The remarkable agreement among computational and experimental studies supports the biophysical relevance of the global fold thus determined for the Aβ(21-30) decapeptide. An important additional observation was that data from the three simulation methods were not entirely identical. This was encouraging because it ruled out the possibility that all the simulations might agree but still be wrong because of the inclusion in each of the same misassumption(s). As an example, only REMD simulations revealed strong hydrogen bonding between Asp23 and other residues within the turn. This observation has stimulated further examination of whether hydrogen bonding may in fact exist within Aβ conformers simulated using DMD and MD or within synthetic peptides in solution. The results thus obtained will strengthen our understanding of Aβ structural biology and improve our simulation algorithms and methods of experimental study. 644 ACCOUNTS OF CHEMICAL RESEARCH / VOL. 39, NO. 9, 2006

5. Summary In hydro, in vacuo, and in silico methods have been integrated into a coordinated program to understand Aβ self-assembly. The integration allows study of phenomena within broad structural and temporal regimes. In hydro experiments reveal relatively gross, population-average features of Aβ monomer folding and oligomerization. These include the roles of turns in nucleating monomer folding and of the C-terminus in mediating oligomerization. IMS can identify/quantify specific oligomer types and produce thermodynamic information about oligomer association. Ab initio in silico procedures, constrained by the experimental results, produce biophysically relevant models of monomer and oligomer structure, reveal atomic contacts, elucidate the temporal (thermo)dynamics of folding and self-association, and allow virtual study of milieu-dependent (e.g., membrane or cytoplasm) folding events. Each discipline informs and validates the others, as well as stimulates new experimental and computational questions. Importantly, the paradigm supports studies of other pathologic proteins and can be applied directly in experimental and computational drug discovery. Strict page limits preclude citation of a large body of excellent work by colleagues in the fields discussed. We acknowledge these efforts here. This work was supported by NIH Grants NS38328, NS44147, AG18921, and AG027818 (D.B.T.), and AG023661 (H.E.S.), NSF Career Award No. 0133504 (J.-E.S.), NSF Grants CHE-0140215 and CHE-0503728 (M.T.B.), and the generosity of the Foundation for Neurologic Diseases (D.B.T.), the A. P. Sloan Foundation (J.E.S.), the David and Lucile Packard Foundation (J.-E.S.), the Alzheimer’s Association (D.B.T. and H.E.S.), and Mr. Stephen Bechtel, Jr. (H.E.S.).

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